Bradyrhizobium strains

ABSTRACT

According to the present invention new isolates of  Bradyrhizobium japonicum  have been isolated and possess unique properties. These  Bradyrhizobia  are plant growth-promoting rhizobacterium (PGPR), possess superior tolerance/resistance to desiccation, and enhance the overall performance of leguminous plant growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/716,510 filed Dec. 17, 2012, now allowed, which claims the benefitunder 35 U.S.C. 119 of U.S. provisional application No. 61/576,470 filedDec. 16, 2011, the contents of which are fully incorporated herein byreference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference. Forcomplete information see Table 1.

FIELD OF THE INVENTION

The present invention relates to isolated Bradyrhizobium bacteriumhaving enhanced characteristics, including but not limited to, enhanceddesiccation resistance.

BACKGROUND OF THE INVENTION

In order to maintain healthy growth, plants must extract a variety ofelements from the soil in which they grow. These elements includenitrogen and the so-called micro-nutrients (e.g., copper, iron andzinc), but many soils are deficient in such elements or they containthem only in forms which cannot be readily taken up by plants (it isgenerally believed that essential elements cannot be readily taken up byplants unless they are present in dissolved form in the soil). Nitrogenis an essential element for most plants as it plays a role in thesynthesis of amino acids, proteins, nucleotides, nucleic acids,chlorophyll, co-enzymes and in the overall growth and health of theplant. To counteract such deficiencies, sources of the deficientelements are commonly applied to soils in order to improve growth ratesand yields obtained from crop plants. For example, nitrate and/orammonium are often added to soil to counteract a lack of availablenitrogen.

In the field of crop science, it is well known that many cultivatedcrops require that the soil provide relatively large amounts of nitrogento the plant. The notable exceptions to those plants requiring nitrogenvia the soil are plants from the legume family.

Specifically, leguminous plants are unique from non-leguminous plants intheir ability to fix atmospheric nitrogen into ammonia. The ability tofix atmospheric nitrogen into a useable nitrogen source for the plantobviates the need for the plant to obtain nitrogen from the soil.Nitrogen fixation, however, requires a symbiotic relationship betweenthe leguminous plant and native bacterial within the soil. One of themost extensively studied partners in this symbiotic relationship isbacteria belonging to the genus Bradyrhizobium or Rhizobium. Gresshoff,P. (1999). Identification of Plant Genes Involved in Plant-MicrobeInteractions. Stacey, G. & Keen, T. (Ed.), Plant-Microbe Interactions(4th ed.) (Ch. 6). St. Paul: APS Press.

Symbiosis is generally achieved through an exchange of complexbidirectional signaling between the plant and the microbe and themicrobe and the plant. Typically, plant factors, such as flavonoids andflavonoid like substances, induce colonization of the bacteria into theroot nodule of the leguminous plant. (Gresshoff, 1999). Once thebacteria have colonized the root nodule, the bacteria effectmorphological changes in the plant, namely root hair curling and thedevelopment of a new root organ—the nodule. (Gresshoff, 1999). Thenodule permits the establishment of a new physiological environment forthe nodule inducing bacteria to differentiate into a nitrogen-fixingendosymbiont, or bacteriod, for the colonized plant. (Gresshoff, 1999).

In order to assist with the symbiotic exchange of bi-directionalsignaling between the plant and microbe, bacteria, such as Bradyrhizobiasp., are often coated on a seed. To prolong the viability of the microbeon the seed, it is desirable that the microbe be tolerant to desiccationand dry environmental conditions generally.

There remains a need for microbes with enhanced desiccation resistance.

SUMMARY OF THE INVENTION

Described herein are novel bacterial strains having enhanced desiccationresistance, especially when the novel strains are compared to itsparental strain, e.g., Bradyrhizobium sp., parental strain USDA 532C.The inventors have isolated and tested a significant number of bacterialstrains for their desiccation resistance properties.

As disclosed throughout, the isolated strains are strains of the genusBradyrhizobium spp. In particular, the isolated strains are strains ofBradyrhizobium japonicum. Even more particularly, the isolated strainsare isolated Bradyrhizobium japonicum strains selected from the groupconsisting of:

the strain having the deposit accession number NRRL B-50608;

the strain having the deposit accession number NRRL B-50609;

the strain having the deposit accession number NRRL B-50610;

the strain having the deposit accession number NRRL B-50611;

the strain having the deposit accession number NRRL B-50612, or acombination of at least two or more of the above deposited strains.

Also described herein are compositions comprising a carrier and one ormore of the bacterial strains described herein. In an embodiment, thecomposition comprises one or more plant signal molecules. In oneembodiment, the composition comprises at least onelipo-chitooligosaccharide (LCO). In another embodiment the compositioncomprises at least one chitooligosaccharide (CO). In still anotherembodiment, the composition comprises at least one flavonoid. In stillyet another embodiment, the composition comprises jasmonic acid or aderivative thereof. In another embodiment, the composition compriseslinoleic acid or a derivative thereof. In yet another embodiment, thecomposition comprises linolenic acid or a derivative thereof. In stillyet another embodiment, the composition comprises a karrikin.

Further described herein is a method for enhancing the growth of a plantor plant part comprising contacting a plant or plant part with one ormore with one or more of the bacterial strains described herein. Themethod comprises introducing into the soil an inoculum of one or more ofthe bacterial strains described herein. In another embodiment, themethod comprises introducing into the soil an inoculum of one or more ofthe bacterial strains as a seed coating.

Also described herein is a method for enhancing nitrogen fixation in aplant(s) comprising growing a plant(s) in a soil that contains a one ormore of the bacterial strains described herein. In one embodiment, theplant(s) is a leguminous plant(s), non-leguminous plant(s), orcombinations thereof. In another embodiment, the plant is a plantselected from the group consisting of soybean, bean, alfalfa, clover,corn, lettuce tomatoes, potatoes, cucumbers, and combinations thereof.

Further described herein are seeds coated with the bacterial strainsdescribed herein.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graphical representation of the preliminary screening ofdesiccation resistant mutants compared when compared to the desiccationresistance of parental strain USDA 532C.

FIG. 2 is a graphical representation of the second screening ofdesiccation resistant mutants compared when compared to the desiccationresistance of parental strain USDA 532C.

FIG. 3 is a graphical representation of the third screening ofdesiccation resistant mutants compared when compared to the desiccationresistance of parental strain USDA 532C.

FIG. 4 is a graphical representation of the fourth screening ofdesiccation resistant mutants compared when compared to the desiccationresistance of parental strain USDA 532C.

FIG. 5 is a bar graph representation of the desiccation resistance ofthe selected desiccation resistant mutants compared when compared to thedesiccation resistance of parental strain USDA 532C at zero (0) andfourteen (14) days.

FIG. 6 is a bar graph representation of the desiccation resistance ofthe selected desiccation resistant mutants compared when compared to thedesiccation resistance of parental strain USDA 532C at seven (7) andfourteen (14) days.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed bacterial strains have been isolated and tested for theirability to solubilize phosphorous. This is described in detail in the“Examples” section provided below. The disclosed embodiments furtherrelate to compositions, seed coatings, methods for increasing theavailability of phosphorus for plant uptake from soil, and methods forincreasing the phosphorus uptake in plants comprising growing the plantsin a soil containing a phosphorus source.

DEFINITIONS

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

As used herein, the term “biologically pure culture” is intended to meana culture essentially free from biological contamination and having agenetic uniformity such that different subcultures taken therefrom willdisplay substantially identical genotypes and phenotypes (e.g., cultureshave a purity of at least 60%, of at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, up to 100% pure).

As used herein, the term “isolate, isolates, isolating, and/or isolated,etc.” is intended to mean that the referenced material is removed fromthe environment in which it is normally found.

As used herein, the term “inoculum” is intended to mean any form ofbacterial cells, or spores, which is capable of propagating on or in thesoil when the conditions of temperature, moisture, etc., are favorablefor bacterial growth.

As used herein, the terms “spore” has its normal meaning which is wellknown and understood by those of skill in the art and generally refersto a microorganism in its dormant, protected state.

As used herein, the term “source” of a particular element is intended tomean a compound of that element which, at least in the soil conditionsunder consideration, does not make the element fully available for plantuptake.

As used herein, the terms “effective amount”, “effective concentration”,or “effective dosage” is intended to mean the amount, concentration, ordosage of the one or more bacterial isolates sufficient to cause anincrease in the growth of a plant or plant part or an increase innitrogen fixation. The actual effective dosage in absolute value dependson factors including, but not limited to, the size (e.g., the area, thetotal acreage, etc.) of the land for application with the bacterialisolates, synergistic or antagonistic interactions between the otheractive or inert ingredients which may increase or reduce activity of thebacterial isolates, and the stability of the bacterial isolates incompositions and/or as seed treatments. The “effective amount”,“effective concentration”, or “effective dosage” of the bacterialcomposition may be determined, e.g., by a routine dose responseexperiment.

As used herein, the terms “carrier” or “agronomically acceptablecarrier” are intended to refer to any material which can be used todeliver the actives (e.g., a bacterial strain) to a seed, soil, plant orplant part.

As used herein, the term “soil-compatible carrier” is intended to referto any material which can be added to a soil without causing/having anadverse effect on plant growth, soil structure, soil drainage, or thelike.

As used herein, the term “seed-compatible carrier” is intended to referto any material which can be added to a seed without causing/having anadverse effect on the seed, the plant that grows from the seed, seedgermination, or the like.

As used herein, the term “agriculturally beneficial ingredient(s)” isintended to mean any agent or combination of agents capable of causingor providing a beneficial and/or useful effect in agriculture.

As used herein, “at least one biologically active ingredient” isintended to mean biologically active ingredients (e.g., signalmolecules, other microorganisms, etc.) other than the one or morebacterial isolates described herein.

As used herein, the term “desiccation” is intended to mean a state ofextreme dryness, e.g., conditions without moisture and/or water. Theterms “desiccation resistance” and/or “desiccation tolerance” areintended to encompass the ability of an organism to withstand and/orsurvive and/or endure conditions of extreme dryness.

As used herein, terms “nitrogen fixation”, “fixation of atmosphericnitrogen”, or “nitrogen fixing”, etc. are intended to encompassbiological processes in which molecular nitrogen or nitrogen in theatmosphere is converted into one or more nitrogenous (N) compounds,including but not limited to, ammonia, ammonium salts, urea, andnitrates.

As used herein, the term “nitrogen fixing organism” is intended to referto any organism (e.g., diazotrophs) capable of converting molecularnitrogen or nitrogen in the atmosphere into one or more nitrogenous (N)compounds, including but not limited to, ammonia, ammonium salts, urea,and nitrates.

As used herein, terms “phosphate solubilization”, or “phosphatesolubilizing”, etc. are intended to mean the conversion of insolublephosphate (e.g., rock phosphate, etc.) into a soluble phosphate form.

As used herein, the term “phosphate solubilizing organism” is intendedto refer to any organism capable of converting insoluble phosphate intoa soluble phosphate form.

As used herein, the terms “plant(s)” and “plant part(s)” are intended torefer to all plants and plant populations such as desired and undesiredwild plants or crop plants (including naturally occurring crop plants).Crop plants can be plants, which can be obtained by conventional plantbreeding and optimization methods or by biotechnological and geneticengineering methods or by combinations of these methods, including thetransgenic plants and including the plant cultivars protectable or notprotectable by plant breeders' rights. Plant parts are to be understoodas meaning all parts and organs of plants above and below the ground,such as shoot, leaf, flower and root, examples which may be mentionedbeing leaves, needles, stalks, stems, flowers, fruit bodies, fruits,seeds, roots, nodules, tubers, and rhizomes. The plant parts alsoinclude harvested material and vegetative and generative propagationmaterial (e.g., cuttings, tubers, rhizomes, off-shoots and seeds, etc.).

As used herein, the term “nodule” is intended to include, but not belimited to, determinate nodules, indeterminate nodules, or a combinationthereof. Examples of determinate nodules and indeterminate nodules arewell known in the art and described in Denison, R. F., 2000, The Amer.Naturalist. 156 (6): 567-576. Determinate nodules are found on Glycine,Lotus, or Phaseolus species and are round and spherical in shape.(Denison, 2000) Determinate nodules grow only for a limited period oftime—typically a few weeks. (Denison, 2000) In contrast to determinatenodules, indeterminate nodules are found on Medicago, Trifolium, andPisium species, have an elongated shape and grow continuously. (Denison,2000) The term “nodule occupancy” is a term known in the art. McDermottT. R. & Graham, P. H., Appl. and Environ. Microbiol. 55(10): 2493-2498.It is well known in the art that, notwithstanding the rare exception, asingle nodule will contain only one bacterial strain. Johnston, A. W.B., et al., 1974, J. Gen. Microbiol. 87: 343-350; Dunham, D. H. &Baldwin, I. L., 1931, Soil Science 32: 235-249; Johnson, H. W., et al.,1963, Agrono. J. 55: 269-271; Dudman, W. F. & Brockwell, J., 1968, J.Agricul. Res. 19: 739-747; Nicol, H. & Thorton, H. G., 1941, Proc. Roy.Soc. B 130, 32-59; Hughes, D. Q., & Vincent, J. M., 1942, Proc. of theLinnenan Soc. of New South Wales 67: 142-152; and Vincent, J. M. &Waters, L. M., 1953, J. Gen. Microbiol. 9: 357-370.

As used herein, term “enhanced plant growth” is intended to refer toincreased plant yield (e.g., increased biomass, increased fruit number,or a combination thereof as measured by bushels per acre), increasedroot number, increased root mass, increased root volume, increased leafarea, increased plant stand, increased plant vigor, increased weight ofa plant (e.g. total dry weight of a plant or plant part, total freshweight or a plant or plant part, etc.), or combinations thereof.

As used herein, “enhanced competitiveness” and/or “enhanced nodulation”is defined to mean bacterial strain(s) possessing a percent noduleoccupancy, e.g. at least 50%, at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, up to100% nodule occupancy.

As used herein, the term “temperature tolerance” is intended to mean therange of temperatures at which a bacterial strain(s) are able to grow,e.g., the maximum and minimum temperatures at which a bacterial straincan grow.

As used herein, the term “commercially available strain(s)” is intendedto mean commercially available bacterial strains, e.g., USDA 532C, USDA110, USDA 123, USDA 127, USDA 129, etc. Cregan, P. B., et al., 1989,Appl. and Enviro. Microbiol. 55 (10): 2532-2536.

As used herein, the term “micronutrient(s)” is intended to refer tonutrients which are needed for plant growth, plant health, and/or plantdevelopment.

As used herein, the term “biostimulant(s)” is intended to refer to anysubstance capable of enhancing metabolic or physiological processeswithin plants and soils.

As used herein, the term “wetting agent(s)” is intended to refer to anysubstance capable of lowering and/or reducing the surface tension ofwater.

Strains

In one embodiment, the isolated strain(s) described herein is a nitrogenfixing bacterial strain(s). In another embodiment, the strain(s) is aBradyrhizobum sp. strain(s). In a further aspect, the strain is derivedfrom a strain of Bradyrhizobium, including but not limited to a strainselected from the group consisting of Bradyrhizobium bete,Bradyrhizobium canariense, Bradyrhizobium elkanii, Bradyrhizobiumiriomotense, Bradyrhizobium japonicum, Bradyrhizobium jicamae,Bradyrhizobium liaoningense, Bradyrhizobium pachyrhizi, andBradyrhizobium yuanmingense. In yet another embodiment, the strain(s) isa Bradyrhizobum japonicum strain(s).

In yet another embodiment, the isolated strain(s) is a strain(s) ofBradyrhizobium sp. having enhanced/increased bacterial survival rate ina substantially moisture free environment when the survival rate of theisolated Bradyrhizobium strain(s) is compared to the survival rate of aparental strain(s), e.g., parental strain Bradyrhizobium japonicum USDA532C, over a period of time, e.g., at least 1 day, at least 2 days, atleast 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, atleast 1 month, at least 2 months, at least 3 months, at least 4 months,at least 5 months, at least 6 months, at least 1 year or more.

In still another embodiment, the isolated strain(s) is a strain(s) ofBradyrhizobium sp. having an enhanced/increased survival rate in asubstantially moisture free environment, wherein an increased survivalrate in a substantially moisture free environment includes an increasedbacterial survival rate in an environment that is at least 70% moisturefree, e.g., at least 75%, at least 80%, at least 85%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, up toa 100% moisture free environment, when the survival rate of the isolatedBradyrhizobium strain(s) is compared to the survival rate of a parentalstrain(s), e.g., parental strain Bradyrhizobium japonicum USDA 532C.

In another embodiment the isolated strain(s) is a strain(s) ofBradyrhizobium sp. having the following enhanced/superiorcharacteristics when compared to commercially available strains, e.g.,commercial strain Bradyrhizobium japonicum USDA 532C, whereinenhanced/superior characteristics include, but are not limited to:

a. enhanced competitiveness for colonizing a soybean plant; and

b. enhanced effectiveness at promoting soybean plant growth.

In still another embodiment, the isolated strain(s) is a strain(s) ofBradyrhizobium sp. having enhanced/superior competitiveness forcolonizing a plant. In yet another embodiment, the isolated strain(s) isa strain(s) of Bradyrhizobium sp. having enhanced/superior effectivenessat promoting plant growth. In still another embodiment, the isolatedstrain(s) is a strain(s) of Bradyrhizobium sp. having enhanced/superiorcompetitiveness for colonizing a plant and enhanced/superioreffectiveness at promoting plant growth.

In yet another aspect of the present invention, the isolated strain(s)is a strain(s) of Bradyrhizobium sp. having enhanced/superiortemperature tolerance.

In still another aspect of the present invention, the isolated strain(s)is a strain(s) of Bradyrhizobium sp. having natural resistance toglyphosate.

In still another embodiment, the strains are Bradyrhizobum japonicumstrains selected from the group consisting of:

the strain having the deposit accession number NRRL B-50608;

the strain having the deposit accession number NRRL B-50609;

the strain having the deposit accession number NRRL B-50610;

the strain having the deposit accession number NRRL B-50611; and

the strain having the deposit accession number NRRL B-50612.

In a particular embodiment, the strain(s) may be one or more of theabove mentioned deposited strains (e.g., including at least two of theabove strains, at least three of the above strains, at least four of theabove strains, up to and including all of the above strains).

In an embodiment, the strain is the strain having the deposit accessionNRRL B-50608. In an embodiment, the strain is the strain having thedeposit accession number NRRL B-50609. In an embodiment, the strain isthe strain having the deposit accession number NRRL B-50610. In anembodiment, the strain is the strain having the deposit accession numberNRRL B-50611. In an embodiment, the strain is the strain having thedeposit accession number NRRL B-50612.

In another embodiment, the bacterial culture(s) hasproperties/characteristics identical to at least one of the depositedstrains or a combination of at least two of the above deposited strains,including more than two, such as, at least three of the above strains,at least four of the above strains, up to and including all of the abovestrains. Properties/characteristics of the bacterial culture include,but are not limited to, bacterial strains having enhanced and/orsuperior resistance to desiccation. In still another embodiment, thestrain(s) is a strain(s) of Bradyrhizobium having enhanced and/orsuperior desiccation resistance when the desiccation resistance iscompared to the desiccation resistance and/or tolerance of a parentalstrain(s) of bacteria, e.g., parental strain Bradyrhizobium japonicumUSDA 532C.

In another aspect, the isolated bacterial strain(s) of the presentinvention includes strain(s) that are closely related to any of theabove strains on the basis of 16S rDNA sequence identity. SeeStackebrandt E, et al., “Report of the ad hoc committee for there-evaluation of the species definition in bacteriology,” Int J SystEvol Microbiol. 52(3):1043-7 (2002) regarding use of 16S rDNA sequenceidentity for determining relatedness in bacteria. In an embodiment, theat least one strain is at least 95% identical to any of the abovestrains on the basis of 16S rDNA sequence identity, at least 96%identical to any of the above strains on the basis of 16S rDNA sequenceidentity, at least 97% identical to any of the above strains on thebasis of 16S rDNA sequence identity, at least 98% to any of the abovestrains on the basis of 16S rDNA sequence identity, at least 98.5%identical to any of the above strains on the basis of 16S rDNA sequenceidentity, at least 99% identical to any of the above strains on thebasis of 16S rDNA sequence identity or at least 99.5% to any of theabove strains on the basis of 16S rDNA sequence identity.

The Bradyrhizobium bacterium described herein, and in particular, thestrains having deposit accession numbers NRRL B-50608, NRRL B-50609,NRRL B-50610, NRRL B-50611, and NRRL B-50612, can be grown according tomethods known in the art.

The resulting material may be used directly in a composition, as a seedtreatment, or the spores may be harvested, concentrated bycentrifugation, formulated, and then dried using air drying, freezedrying, or fluid bed drying techniques (Friesen T., Hill G., Pugsley T.,Holloway G., and Zimmerman D. 2005, Experimental determination ofviability loss of Penicillium bilaiae conidia during convectiveair-drying Appl Microbiol Biotechnol 68: 397-404) to produce a wettablepowder.

Above mentioned deposited strains were deposited on Nov. 30, 2011, asindicated in more details below in the “Materials & Methods”-section,under terms of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure atAmerican Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va.20108, USA.

Compositions:

In another aspect, the invention relates to a composition comprising acarrier and an inoculum of one or more of the deposited strains (eitherspore form or strains in a vegetative state) described herein. Incertain embodiments, the composition may be in the form of a liquid, aslurry, a solid, or a powder (wettable powder or dry powder). In anotherembodiment, the composition may be in the form of a seed coating.Compositions in liquid, slurry, or powder (e.g., wettable powder) formmay be suitable for coating seeds. When used to coat seeds, thecomposition may be applied to the seeds and allowed to dry. Inembodiments wherein the composition is a powder (e.g., a wettablepowder), a liquid, such as water, may need to be added to the powderbefore application to a seed. Example of yet other carriers includemoistened bran, dried, sieved and applied to seeds prior coated with anadhesive, e.g., gum arabic.

Carriers:

The carriers described herein will allow the deposited bacterialstrain(s) to remain efficacious (e.g., capable of fixing nitrogen) andviable once formulated. Non-limiting examples of carriers describedherein include liquids, slurries, or solids (including wettable powdersor dry powders). In an embodiment, the carrier is a soil compatiblecarrier as described herein.

In one embodiment, the carrier is a liquid carrier. Non-limitingexamples of liquids useful as carriers for the compositions disclosedherein include water, an aqueous solution, or a non-aqueous solution. Inone embodiment, the carrier is water. In another embodiment the carrieris an aqueous solution, such as sugar water. In another embodiment, thecarrier is a non-aqueous solution. If a liquid carrier is used, theliquid (e.g., water) carrier may further include growth media to culturethe deposited bacterial strains. Non-limiting examples of suitablegrowth media for the deposited bacterial strains includearabinose-gluconate (AG), yeast extract mannitol (YEM), G16 media, orany media known to those skilled in the art to be compatible with,and/or provide growth nutrients to the deposited bacterial strains.

In another embodiment, the carrier is a slurry. In an embodiment, theslurry may comprise a sticking agent, a liquid, or a combinationthereof. It is envisioned that the sticking agent can be any agentcapable of sticking the inoculum (e.g., one or more of the depositedstrains) to a substrate of interest (e.g., a seed). Non-limitingexamples of sticking agents include alginate, mineral oil, syrup, gumarabic, honey, methyl cellulose, milk, wallpaper paste, and combinationsthereof. Non-limiting examples of liquids appropriate for a slurryinclude water or sugar water.

In another embodiment, the carrier is a solid. In a particularembodiment the solid is a powder. In one embodiment the powder is awettable powder. In another embodiment, the powder is a dry powder. Inanother embodiment, the solid is a granule. Non-limiting examples ofsolids useful as carriers for the compositions disclosed herein includepeat, wheat, wheat chaff, ground wheat straw, bran (e.g., moistenedbran, non-moistened bran), vermiculite, cellulose, starch, soil(pasteurized or unpasteurized), gypsum, talc, clays (e.g., kaolin,bentonite, montmorillonite), and silica gels.

Optional Agriculturally Beneficial Ingredients:

The compositions disclosed herein may comprise one or more optionalingredients. Non-limiting examples of optional ingredients include oneor more biologically active ingredients, micronutrients, biostimulants,preservatives, polymers, wetting agents, surfactants, or combinationsthereof.

Biologically Active Ingredient(s):

The compositions described herein may optionally include one or morebiologically active ingredients as described herein. Non-limitingexamples of biologically active ingredients include signal molecules(e.g., lipo-chitooligosaccharides (LCO), chitooligosaccharides (CO),chitinous compounds, flavonoids, jasmonic acid or derivatives thereof,linoleic acid or derivatives thereof, linolenic acid or derivativesthereof, kerrikins, etc.) and beneficial microorganisms (e.g., Rhizobiumspp., Bradyrhizobium spp., Sinorhizobium spp., Azorhizobium spp., etc.).

Signal Molecule(s):

In an embodiment, the compositions described herein include one or moresignal molecules. In one embodiment, the one or more signal moleculesare one or more LCOs. In another embodiment, the one or more signalmolecules are one or more chitinous compounds. In still anotherembodiment, the one or more signal molecules are one or more COs. In yetanother embodiment, the one or more signal molecules are one or moreflavonoids or derivatives thereof. In still yet another embodiment, theone or more signal molecules are one or more non-flavonoid nod geneinducers (e.g., jasmonic acid, linoleic acid, linolenic acid, andderivatives thereof). In still yet another embodiment, the one or moresignal molecules are one or more karrikins or derivatives thereof. Instill another embodiment, the one or more signal molecules are one ormore LCOs, one or more chitinous compounds, one or more COs, one or moreflavonoids and derivatives thereof, one or more non-flavonoid nod geneinducers and derivatives thereof, one or more karrikins and derivativesthereof, or any signal molecule combination thereof.

LCOs:

Lipo-chitooligosaccharide compounds (LCDs), also known in the art assymbiotic Nod signals or Nod factors, consist of an oligosaccharidebackbone of β-1,4-linked N-acetyl-D-glucosamine (“GlcNAc”) residues withan N-linked fatty acyl chain condensed at the non-reducing end. LCO'sdiffer in the number of GlcNAc residues in the backbone, in the lengthand degree of saturation of the fatty acyl chain, and in thesubstitutions of reducing and non-reducing sugar residues. An example ofan LCO is presented below as formula I:

in which:

G is a hexosamine which can be substituted, for example, by an acetylgroup on the nitrogen, a sulfate group, an acetyl group and/or an ethergroup on an oxygen,

R₁, R₂, R₃, R₅, R₆ and R₇, which may be identical or different,represent H, CH₃ CO—, C_(x) H_(y) CO— where x is an integer between 0and 17, and y is an integer between 1 and 35, or any other acyl groupsuch as for example a carbamyl,

R₄ represents a mono-, di- or triunsaturated aliphatic chain containingat least 12 carbon atoms, and n is an integer between 1 and 4.

LCOs may be obtained (isolated and/or purified) from bacteria such asRhizobia, e.g., Rhizobium spp., Bradyrhizobium spp., Sinorhizobium spp.and Azorhizobium spp. LCO structure is characteristic for each suchbacterial species, and each strain may produce multiple LCO's withdifferent structures. For example, specific LCOs from S. meliloti havealso been described in U.S. Pat. No. 5,549,718 as having the formula II:

in which R represents H or CH₃CO— and n is equal to 2 or 3.

Even more specific LCOs include NodRM, NodRM-1, NodRM-3. When acetylated(the R═CH₃ CO—), they become AcNodRM-1, and AcNodRM-3, respectively(U.S. Pat. No. 5,545,718).

LCOs from Bradyrhizobium japonicum are described in U.S. Pat. Nos.5,175,149 and 5,321,011. Broadly, they are pentasaccharide phytohormonescomprising methylfucose. A number of these B. japonicum-derived LCOs aredescribed: BjNod-V (C_(18:1)); BjNod-V (A_(C), C_(18:1)), BjNod-V(C_(16:1)); and BjNod-V (A_(C), C_(16:0)), with “V” indicating thepresence of five N-acetylglucosamines; “Ac” an acetylation; the numberfollowing the “C” indicating the number of carbons in the fatty acidside chain; and the number following the “:” the number of double bonds.

LCOs used in compositions of the invention may be obtained (i.e.,isolated and/or purified) from bacterial strains that produce LCO's,such as strains of Azorhizobium, Bradyrhizobium (including B.japonicum), Mesorhizobium, Rhizobium (including R. leguminosarum),Sinorhizobium (including S. meliloti), and bacterial strains geneticallyengineered to produce LCO's.

Also encompassed by the present invention are compositions using LCOsobtained (i.e., isolated and/or purified) from a mycorrhizal fungus,such as fungi of the group Glomerocycota, e.g., Glomus intraradicus. Thestructures of representative LCOs obtained from these fungi aredescribed in WO 2010/049751 and WO 2010/049751 (the LCOs describedtherein also referred to as “Myc factors”).

Further encompassed by compositions of the present invention is use ofsynthetic LCO compounds, such as those described in WO 2005/063784, andrecombinant LCO's produced through genetic engineering. The basic,naturally occurring LCO structure may contain modifications orsubstitutions found in naturally occurring LCO's, such as thosedescribed in Spaink, Crit. Rev. Plant Sci. 54:257-288 (2000) andD'Haeze, et al., Glycobiology 12:79R-105R (2002). Precursoroligosaccharide molecules (COs, which as described below, are alsouseful as plant signal molecules in the present invention) for theconstruction of LCOs may also be synthesized by genetically engineeredorganisms, e.g., as in Samain, et al., Carb. Res. 302:35-42 (1997);Samain, et al., J. Biotechnol. 72:33-47 (1999).

LCO's may be utilized in various forms of purity and may be used aloneor in the form of a culture of LCO-producing bacteria or fungi. Methodsto provide substantially pure LCO's include simply removing themicrobial cells from a mixture of LCOs and the microbe, or continuing toisolate and purify the LCO molecules through LCO solvent phaseseparation followed by HPLC chromatography as described, for example, inU.S. Pat. No. 5,549,718. Purification can be enhanced by repeated HPLC,and the purified LCO molecules can be freeze-dried for long-termstorage.

COs:

Chitooligosaccharides (COs) are known in the art as β-1-4 linked N actylglucosamine structures identified as chitin oligomers, also asN-acetylchitooligosaccharides. CO's have unique and different side chaindecorations which make them different from chitin molecules[(C₈H₁₃NO₅)n, CAS No. 1398-61-4], and chitosan molecules [(C₅H₁₁NO₄)n,CAS No. 9012-76-4]. Representative literature describing the structureand production of COs is as follows: Van der Hoist, et al., CurrentOpinion in Structural Biology, 11:608-616 (2001); Robina, et al.,Tetrahedron 58:521-530 (2002); Hanel, et al., Planta 232:787-806 (2010);Rouge, et al. Chapter 27, “The Molecular Immunology of ComplexCarbohydrates” in Advances in Experimental Medicine and Biology,Springer Science; Wan, et al., Plant Cell 21:1053-69 (2009);PCT/F100/00803 (Sep. 21, 2000); and Demont-Caulet, et al., PlantPhysiol. 120(1):83-92 (1999). The COs may be synthetic or recombinant.Methods for preparation of recombinant COs are known in the art. See,e.g., Samain, et al. (supra.); Cottaz, et al., Meth. Eng. 7(4):311-7(2005) and Samain, et al., J. Biotechnol. 72:33-47 (1999).

Chitinous Compounds:

Chitins and chitosans, which are major components of the cell walls offungi and the exoskeletons of insects and crustaceans, are also composedof GlcNAc residues. Chitinous compounds include chitin, (IUPAC:N-[5-[[3-acetylamino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]methoxymethyl]-2-[[5-acetylamino-4,6-dihydroxy-2-(hydroxymethyl)oxan-3-yl]methoxymethyl]-4-hydroxy-6-(hydroxymethyl)oxan-3-ys]ethanamide),and chitosan, (IUPAC:5-amino-6-[5-amino-6-[5-amino-4,6-dihydroxy-2(hydroxymethyl)oxan-3-yl]oxy-4-hydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-2(hydroxymethyl)oxane-3,4-diol).

These compounds may be obtained commercially, e.g., from Sigma-Aldrich,or prepared from insects, crustacean shells, or fungal cell walls.Methods for the preparation of chitin and chitosan are known in the art,and have been described, for example, in U.S. Pat. No. 4,536,207(preparation from crustacean shells), Pochanavanich, et al., Lett. Appl.Microbiol. 35:17-21 (2002) (preparation from fungal cell walls), andU.S. Pat. No. 5,965,545 (preparation from crab shells and hydrolysis ofcommercial chitosan). Deacetylated chitins and chitosans may be obtainedthat range from less than 35% to greater than 90% deacetylation, andcover a broad spectrum of molecular weights, e.g., low molecular weightchitosan oligomers of less than 15 kD and chitin oligomers of 0.5 to 2kD; “practical grade” chitosan with a molecular weight of about 15 kD;and high molecular weight chitosan of up to 70 kD. Chitin and chitosancompositions formulated for seed treatment are also commerciallyavailable. Commercial products include, for example, ELEXA® (PlantDefense Boosters, Inc.) and BEYOND™ (Agrihouse, Inc.).

Flavonoids:

Flavonoids are phenolic compounds having the general structure of twoaromatic rings connected by a three-carbon bridge. Flavonoids areproduced by plants and have many functions, e.g., as beneficialsignaling molecules, and as protection against insects, animals, fungiand bacteria. Classes of flavonoids include chalcones, anthocyanidins,coumarins, flavones, flavanols, flavonols, flavanones, and isoflavones.See, Jain, et al., J. Plant Biochem. & Biotechnol. 11:1-10 (2002); Shaw,et al., Environmental Microbiol. 11:1867-80 (2006).

Representative flavonoids that may be useful in compositions of thepresent invention include luteolin, apigenin, tangeritin, quercetin,kaempferol, myricetin, fisetin, isorhamnetin, pachypodol, rhamnazin,hesperetin, naringenin, formononetin, eriodictyol, homoeriodictyol,taxifolin, dihydroquercetin, dihydrokaempferol, genistein, daidzein,glycitein, catechin, gallocatechin, catechin 3-gallate, gallocatechin3-gallate, epicatechin, epigallocatechin, epicatechin 3-gallate,epigallocatechin 3-gallate, cyaniding, delphinidin, malvidin,pelargonidin, peonidin, petunidin, or derivatives thereof. Flavonoidcompounds are commercially available, e.g., from Natland InternationalCorp., Research Triangle Park, N.C.; MP Biomedicals, Irvine, Calif.; LCLaboratories, Woburn Mass. Flavonoid compounds may be isolated fromplants or seeds, e.g., as described in U.S. Pat. Nos. 5,702,752;5,990,291; and 6,146,668. Flavonoid compounds may also be produced bygenetically engineered organisms, such as yeast, as described inRalston, et al., Plant Physiology 137:1375-88 (2005).

Non-Flavonoid Nod-Gene Inducer(s):

Jasmonic acid (JA, [1R-[1α,2β(Z)]]-3-oxo-2-(pentenyl)cyclopentaneaceticacid) and its derivatives, linoleic acid ((Z,Z)-9,12-Octadecadienoicacid) and its derivatives, and linolenic acid((Z,Z,Z)-9,12,15-octadecatrienoic acid) and its derivatives, may also beused in compositions of the present invention. Jasmonic acid and itsmethyl ester, methyl jasmonate (MeJA), collectively known as jasmonates,are octadecanoid-based compounds that occur naturally in plants.Jasmonic acid is produced by the roots of wheat seedlings, and by fungalmicroorganisms such as Botryodiplodia theobromae and Gibbrellafujikuroi, yeast (Saccharomyces cerevisiae), and pathogenic andnon-pathogenic strains of Escherichia coli. Linoleic acid and linolenicacid are produced in the course of the biosynthesis of jasmonic acid.Jasmonates, linoleic acid and linoleic acid (and their derivatives) arereported to be inducers of nod gene expression or LCO production byrhizobacteria. See, e.g., Mabood, Fazli, Jasmonates induce theexpression of nod genes in Bradyrhizobium japonicum, May 17, 2001; andMabood, Fazli, “Linoleic and linolenic acid induce the expression of nodgenes in Bradyrhizobium japonicum,” USDA 3, May 17, 2001.

Useful derivatives of linoleic acid, linolenic acid, and jasmonic acidthat may be useful in compositions of the present invention includeesters, amides, glycosides and salts. Representative esters arecompounds in which the carboxyl group of linoleic acid, linolenic acid,or jasmonic acid has been replaced with a —COR group, where R is an —OR¹group, in which R¹ is: an alkyl group, such as a C₁-C₈ unbranched orbranched alkyl group, e.g., a methyl, ethyl or propyl group; an alkenylgroup, such as a C₂-C₈ unbranched or branched alkenyl group; an alkynylgroup, such as a C₂-C₈ unbranched or branched alkynyl group; an arylgroup having, for example, 6 to 10 carbon atoms; or a heteroaryl grouphaving, for example, 4 to 9 carbon atoms, wherein the heteroatoms in theheteroaryl group can be, for example, N, O, P, or S. Representativeamides are compounds in which the carboxyl group of linoleic acid,linolenic acid, or jasmonic acid has been replaced with a —COR group,where R is an NR²R³ group, in which R² and R³ are independently:hydrogen; an alkyl group, such as a C₁-C₈ unbranched or branched alkylgroup, e.g., a methyl, ethyl or propyl group; an alkenyl group, such asa C₂-C₈ unbranched or branched alkenyl group; an alkynyl group, such asa C₂-C₈ unbranched or branched alkynyl group; an aryl group having, forexample, 6 to 10 carbon atoms; or a heteroaryl group having, forexample, 4 to 9 carbon atoms, wherein the heteroatoms in the heteroarylgroup can be, for example, N, O, P, or S. Esters may be prepared byknown methods, such as acid-catalyzed nucleophilic addition, wherein thecarboxylic acid is reacted with an alcohol in the presence of acatalytic amount of a mineral acid. Amides may also be prepared by knownmethods, such as by reacting the carboxylic acid with the appropriateamine in the presence of a coupling agent such as dicyclohexylcarbodiimide (DCC), under neutral conditions. Suitable salts of linoleicacid, linolenic acid, and jasmonic acid include e.g., base additionsalts. The bases that may be used as reagents to prepare metabolicallyacceptable base salts of these compounds include those derived fromcations such as alkali metal cations (e.g., potassium and sodium) andalkaline earth metal cations (e.g., calcium and magnesium). These saltsmay be readily prepared by mixing together a solution of linoleic acid,linolenic acid, or jasmonic acid with a solution of the base. The saltmay be precipitated from solution and be collected by filtration or maybe recovered by other means such as by evaporation of the solvent.

Karrikin(s):

Karrikins are vinylogous 4H-pyrones e.g., 2H-furo[2,3-c]pyran-2-onesincluding derivatives and analogues thereof. Examples of these compoundsare represented by the following structure:

wherein; Z is O, S or NR₅; R₁, R₂, R₃, and R₄ are each independently H,alkyl, alkenyl, alkynyl, phenyl, benzyl, hydroxy, hydroxyalkyl, alkoxy,phenyloxy, benzyloxy, CN, COR₆, COOR═, halogen, NR₆R₇, or NO₂; and R₅,R₆, and R₇ are each independently H, alkyl or alkenyl, or a biologicallyacceptable salt thereof. Examples of biologically acceptable salts ofthese compounds may include acid addition salts formed with biologicallyacceptable acids, examples of which include hydrochloride, hydrobromide,sulphate or bisulphate, phosphate or hydrogen phosphate, acetate,benzoate, succinate, fumarate, maleate, lactate, citrate, tartrate,gluconate; methanesulphonate, benzenesulphonate and p-toluenesulphonicacid. Additional biologically acceptable metal salts may include alkalimetal salts, with bases, examples of which include the sodium andpotassium salts. Examples of compounds embraced by the structure andwhich may be suitable for use in the present invention include thefollowing: 3-methyl-2H-furo[2,3-c]pyran-2-one (where R₁═CH₃, R₂, R₃,R₄═H), 2H-furo[2,3-c]pyran-2-one (where R₁, R₂, R₃, R4=H),7-methyl-2H-furo[2,3-c]pyran-2-one (where R₁, R₂, R₄═H, R₃═CH₃),5-methyl-2H-furo[2,3-c]pyran-2-one (where R₁, R₂, R₃═H, R₄═CH₃),3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (where R₁, R₃═CH₃, R₂, R₄═H),3,5-dimethyl-2H-furo[2,3-c]pyran-2-one (where R₁, R₄═CH₃, R₂, R₃═H),3,5,7-trimethyl-2H-furo[2,3-c]pyran-2-one (where R₁, R₃, R₄═CH₃, R₂═H),5-methoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (where R₁═CH₃, R₂,R₃═H, R₄═CH₂OCH₃), 4-bromo-3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (whereR₁, R₃═CH₃, R₂=Br, R₄═H), 3-methylfuro[2,3-c]pyridin-2(3H)-one (whereZ═NH, R₁═CH₃, R₂, R₃, R₄═H), 3,6-dimethylfuro[2,3-c]pyridin-2(6H)-one(where Z═N—CH₃, R₁═CH₃, R₂, R₃, R₄═H). See, U.S. Pat. No. 7,576,213.These molecules are also known as karrikins. See, Halford, “SmokeSignals,” in Chem. Eng. News (Apr. 12, 2010), at pages 37-38 (reportingthat karrikins or butenolides which are contained in smoke act as growthstimulants and spur seed germination after a forest fire, and caninvigorate seeds such as corn, tomatoes, lettuce and onions that hadbeen stored). These molecules are the subject of U.S. Pat. No.7,576,213.

Beneficial Microorganism(s):

In an embodiment, the compositions described herein may comprise one ormore beneficial microorganisms. The one or more beneficialmicroorganisms may have one or more beneficial properties (e.g., produceone or more of the signal molecules described herein, enhance nutrientand water uptake, enhance growth, enhance seed germination, enhanceseedling emergence, break the dormancy or quiescence of a plant, etc.).

In one embodiment, the beneficial microorganism(s) comprise one or morebacteria that produce one or more of the signal molecules describedherein. In still another embodiment, the bacteria are bacteria from thegenera Rhizobium spp. (e.g., R. cellulosilyticum, R. daejeonense, R.etli, R. galegae, R. gallicum, R. giardinii, R. hainanense, R.huautlense, R. indigoferae, R. leguminosarum, R. loessense, R. lupini,R. lusitanum, R. meliloti, R. mongolense, R. miluonense, R. sullae, R.tropici, R. undicola, and/or R. yanglingense), Azorhizobium spp. (e.g.,A. caulinodans and/or A. doebereinerae), Sinorhizobium spp. (e.g., S.abri, S. adhaerens, S. americanum, S. aboris, S. fredii, S. indiaense,S. kostiense, S. kummerowiae, S. medicae, S. meliloti, S. mexicanus, S.morelense, S. saheli, S. terangae, and/or S. xinjiangense),Mesorhizobium spp., (M. albiziae, M. amorphae, M. chacoense, M. ciceri,M. huakuii, M. loti, M. mediterraneum, M. pluifarium, M. septentrionale,M. temperatum, and/or M. tianshanense), and combinations thereof. In aparticular embodiment, the beneficial microorganism is selected from thegroup consisting of R. leguminosarum, R. meliloti, S. meliloti, andcombinations thereofln another embodiment, the beneficial microorganismis R. leguminosarum. In another embodiment, the beneficial microorganismis R. meliloti. In another embodiment, the beneficial microorganism isS. meliloti.

In another embodiment, the one or more beneficial microorganismscomprise one or more phosphate solubilizing microorganisms. Phosphatesolubilizing microorganisms include fungal and bacterial strains. In anembodiment, the phosphate solubilizing microorganism includes speciesfrom a genus selected from the group consisting of Acinetobacter spp.(e.g., Acinetobacter calcoaceticus, etc.), Arthrobacter spp,Arthrobotrys spp. (e.g., Arthrobotrys oligospora, etc.), Aspergillusspp. (e.g., Aspergillus niger, etc.), Azospirillum spp. (e.g.,Azospirillum halopraeferans, etc.), Bacillus spp. (e.g., Bacillusamyloliquefaciens, Bacillus atrophaeus, Bacillus circulans, Bacilluslicheniformis, Bacillus subtilis, etc.), Burkholderia spp. (e.g.,Burkholderia cepacia, Burkholderia vietnamiensis, etc.), Candida spp.(e.g., Candida krissii, etc.), Chryseomonas spp. (e.g., Chryseomonasluteola, etc.), Enterobacter spp. (e.g., Enterobacter aerogenes,Enterobacter asburiae, Enterobacter spp., Enterobacter taylorae, etc.),Eupenicillium spp. (e.g., Eupenicillium parvum, etc.), Exiguobacteriumspp., Klebsiella spp., Kluyvera spp. (e.g., Kluyvera cryocrescens,etc.), Microbacterium spp., Mucor spp. (e.g., Mucor ramosissimus, etc.),Paecilomyces spp. (e.g., Paecilomyces hepialid, Paecilomyces marquandii,etc.), Paenibacillus spp. (e.g., Paenibacillus macerans, Paenibacillusmucilaginosus, etc.), Penicillium spp. (e.g., Penicillium bilaiae(formerly known as Penicillium bilaii), Penicillium albidum, Penicilliumaurantiogriseum, Penicillium chrysogenum, Penicillium citreonigrum,Penicillium citrinum, Penicillium digitatum, Penicillium frequentas,Penicillium fuscum, Penicillium gaestrivorus, Penicillium glabrum,Penicillium griseofulvum, Penicillium implicatum, Penicilliumjanthinellum, Penicillium lilacinum, Penicillium minioluteum,Penicillium montanense, Penicillium nigricans, Penicillium oxalicum,Penicillium pinetorum, Penicillium pinophilum, Penicillium purpurogenum,Penicillium radicans, Penicillium radicum, Penicillium raistrickii,Penicillium rugulosum, Penicillium simplicissimum, Penicillium solitum,Penicillium variabile, Penicillium velutinum, Penicillium viridicatum,Penicillium glaucum, Penicillium fussiporus, and Penicillium expansum,etc.), Pseudomonas spp. (e.g., Pseudomonas corrugate, Pseudomonasfluorescens, Pseudomonas lutea, Pseudomonas poae, Pseudomonas putida,Pseudomonas stutzeri, Pseudomonas trivialis, etc.), Serratia spp. (e.g.,Serratia marcescens, etc.), Stenotrophomonas spp. (e.g.,Stenotrophomonas maltophilia, etc.), Streptomyces spp.,Streptosporangium spp., Swaminathania spp. (e.g., Swaminathaniasalitolerans, etc.), Thiobacillus spp. (e.g., Thiobacillus ferrooxidans,etc.), Torulospora spp. (e.g., Torulospora globosa, etc.), Vibrio spp.(e.g., Vibrio proteolyticus, etc.), Xanthobacter spp. (e.g.,Xanthobacter agilis, etc.), Xanthomonas spp. (e.g., Xanthomonascampestris, etc.), and combinations thereof.

In a particular embodiment, the one or more phosphate solubilizingmicroorganisms is a strain of the fungus Penicillium. In anotherembodiment, the one or more Penicillium species is P. bilaiae, P.gaestrivorus, or combinations thereof.

In another embodiment the beneficial microorganism is one or moremycorrhiza. In particular, the one or more mycorrhiza is anendomycorrhiza (also called vesicular arbuscular mycorrhizas, VAMs,arbuscular mycorrhizas, or AMs), an ectomycorrhiza, or a combinationthereof.

In one embodiment, the one or more mycorrhiza is an endomycorrhiza ofthe phylum Glomeromycota and genera Glomus and Gigaspora. In still afurther embodiment, the endomycorrhiza is a strain of Glomus aggregatum,Glomus brasilianum, Glomus clarum, Glomus deserticola, Glomusetunicatum, Glomus fasciculatum, Glomus intraradices, Glomus monosporum,or Glomus mosseae, Gigaspora margarita, or a combination thereof.

In another embodiment, the one or more mycorrhiza is an ectomycorrhizaof the phylum Basidiomycota, Ascomycota, and Zygomycota. In still yetanother embodiment, the ectomycorrhiza is a strain of Laccaria bicolor,Laccaria laccata, Pisolithus tinctorius, Rhizopogon amylopogon,Rhizopogon fulvigleba, Rhizopogon luteolus, Rhizopogon villosuli,Scleroderma cepa, Scleroderma citrinum, or a combination thereof.

In still another embodiment, the one or more mycorrhiza is an ecroidmycorrhiza, an arbutoid mycorrhiza, or a monotropoid mycorrhiza.Arbuscular and ectomycorrhizas form ericoid mycorrhiza with many plantsbelonging to the order Ericales, while some Ericales form arbutoid andmonotropoid mycorrhizas. All orchids are mycoheterotrophic at some stageduring their lifecycle and form orchid mycorrhizas with a range ofbasidiomycete fungi. In one embodiment, the mycorrhiza may be an ericoidmycorrhiza, preferably of the phylum Ascomycota, such as Hymenoscyphousericae or Oidiodendron sp. In another embodiment, the mycorrhiza alsomay be an arbutoid mycorrhiza, preferably of the phylum Basidiomycota.In yet another embodiment, the mycorrhiza may be a monotripoidmycorrhiza, preferably of the phylum Basidiomycota. In still yet anotherembodiment, the mycorrhiza may be an orchid mycorrhiza, preferably ofthe genus Rhizoctonia.

Micronutrient(s):

In still another embodiment, the compositions described herein maycomprise one or more beneficial micronutrients. Non-limiting examples ofmicronutrients for use in the compositions described herein includevitamins, (e.g., vitamin A, vitamin B complex (i.e., vitamin B₁, vitaminB₂, vitamin B₃, vitamin B₅, vitamin B₆, vitamin B₇, vitamin B₈, vitaminB₉, vitamin B₁₂, choline) vitamin C, vitamin D, vitamin E, vitamin K,carotenoids (α-carotene, β-carotene, cryptoxanthin, lutein, lycopene,zeaxanthin, etc.), macrominerals (e.g., phosphorous, calcium, magnesium,potassium, sodium, iron, etc.), trace minerals (e.g., boron, cobalt,chloride, chromium, copper, fluoride, iodine, iron, manganese,molybdenum, selenium, zinc, etc.), organic acids (e.g., acetic acid,citric acid, lactic acid, malic aclid, taurine, etc.), and combinationsthereof. In a particular embodiment, the compositions may comprisephosphorous, boron, chlorine, copper, iron, manganese, molybdenum, zincor combinations thereof.

In certain embodiments, where the compositions described herein maycomprise phosphorous, it is envisioned that any suitable source ofphosphorous may be provided. In one embodiment, the phosphorus may bederived from a source. In another embodiment, suitable sources ofphosphorous include phosphorous sources capable of solubilization by oneor more microorganisms (e.g., Penicillium bilaiae, etc.).

In one embodiment, the phosphorus may be derived from a rock phosphatesource. In another embodiment the phosphorous may be derived fromfertilizers comprising one or more phosphorous sources. Commerciallyavailable manufactured phosphate fertilizers are of many types. Somecommon ones are those containing rock phosphate, monoammonium phosphate,diammonium phosphate, monocalcium phosphate, super phosphate, triplesuper phosphate, and/or ammonium polyphosphate. All of these fertilizersare produced by chemical processing of insoluble natural rock phosphatesin large scale fertilizer-manufacturing facilities and the product isexpensive. By means of the present invention it is possible to reducethe amount of these fertilizers applied to the soil while stillmaintaining the same amount of phosphorus uptake from the soil.

In still another embodiment, the phosphorous may be derived from anorganic phosphorous source. In a further particular embodiment, thesource of phosphorus may include an organic fertilizer. An organicfertilizer refers to a soil amendment derived from natural sources thatguarantees, at least, the minimum percentages of nitrogen, phosphate,and potash. Non-limiting examples of organic fertilizers include plantand animal by-products, rock powders, seaweed, inoculants, andconditioners. These are often available at garden centers and throughhorticultural supply companies. In particular the organic source ofphosphorus is from bone meal, meat meal, animal manure, compost, sewagesludge, or guano, or combinations thereof.

In still another embodiment, the phosphorous may be derived from acombination of phosphorous sources including, but not limited to, rockphosphate, fertilizers comprising one or more phosphorous sources (e.g.,monoammonium phosphate, diammonium phosphate, monocalcium phosphate,super phosphate, triple super phosphate, ammonium polyphosphate, etc.)one or more organic phosphorous sources, and combinations thereof.

Biostimulant(s):

In one embodiment, the compositions described herein may comprise one ormore beneficial biostimulants. Biostimulants may enhance metabolic orphysiological processes such as respiration, photosynthesis, nucleicacid uptake, ion uptake, nutrient delivery, or a combination thereof.Non-limiting examples of biostimulants include seaweed extracts (e.g.,ascophyllum nodosum), humic acids (e.g., potassium humate), fulvicacids, myo-inositol, glycine, and combinations thereof. In anotherembodiment, the compositions comprise seaweed extracts, humic acids,fulvic acids, myo-inositol, glycine, and combinations thereof.

Polymer(s):

In one embodiment, the compositions described herein may furthercomprise one or more polymers. Non-limiting uses of polymers in theagricultural industry include agrochemical delivery, heavy metalremoval, water retention and/or water delivery, and combinationsthereof. Pouci, et al., Am. J. Agri. & Biol. Sci., 3(1):299-314 (2008).In one embodiment, the one or more polymers is a natural polymer (e.g.,agar, starch, alginate, pectin, cellulose, etc.), a synthetic polymer, abiodegradable polymer (e.g., polycaprolactone, polylactide, poly (vinylalcohol), etc.), or a combination thereof.

For a non-limiting list of polymers useful for the compositionsdescribed herein, see Pouci, et al., Am. J. Agri. & Biol. Sci.,3(1):299-314 (2008). In one embodiment, the compositions describedherein comprise cellulose, cellulose derivatives, methylcellulose,methylcellulose derivatives, starch, agar, alginate, pectin,polyvinylpyrrolidone, and combinations thereof.

Wetting Agent(s):

In one embodiment, the compositions described herein may furthercomprise one or more wetting agents. Wetting agents are commonly used onsoils, particularly hydrophobic soils, to improve the infiltrationand/or penetration of water into a soil. The wetting agent may be anadjuvant, oil, surfactant, buffer, acidifier, or combination thereof. Inan embodiment, the wetting agent is a surfactant. In an embodiment, thewetting agent is one or more nonionic surfactants, one or more anionicsurfactants, or a combination thereof. In yet another embodiment, thewetting agent is one or more nonionic surfactants.

Surfactants suitable for the compositions described herein are providedin the “Surfactants” section.

Surfactant(s):

Surfactants suitable for the compositions described herein may benon-ionic surfactants (e.g., semi-polar and/or anionic and/or cationicand/or zwitterionic). It is envisioned that the surfactant(s) will causeas little harm to the activity of the one or more deposited strainsand/or the one or more beneficial microorganisms as possible. Thesurfactants can wet and emulsify soil(s) and/or dirt(s). It isenvisioned that the surfactants used in described composition have lowtoxicity for the microorganisms contained within the formulation. It isfurther envisioned that the surfactants used in the describedcomposition have a low phytotoxicity (i.e., the degree of toxicity asubstance or combination of substances has on a plant). A singlesurfactant or a blend of several surfactants can be used.

Anionic Surfactants

Anionic surfactants or mixtures of anionic and nonionic surfactants mayalso be used in the compositions. Anionic surfactants are surfactantshaving a hydrophilic moiety in an anionic or negatively charged state inaqueous solution. The compositions described herein may comprise one ormore anionic surfactants. The anionic surfactant(s) may be either watersoluble anionic surfactants, water insoluble anionic surfactants, or acombination of water soluble anionic surfactants and water insolubleanionic surfactants. Non-limiting examples of anionic surfactantsinclude sulfonic acids, sulfuric acid esters, carboxylic acids, andsalts thereof. Non-limiting examples of water soluble anionicsurfactants include alkyl sulfates, alkyl ether sulfates, alkyl amidoether sulfates, alkyl aryl polyether sulfates, alkyl aryl sulfates,alkyl aryl sulfonates, monoglyceride sulfates, alkyl sulfonates, alkylamide sulfonates, alkyl aryl sulfonates, benzene sulfonates, toluenesulfonates, xylene sulfonates, cumene sulfonates, alkyl benzenesulfonates, alkyl diphenyloxide sulfonate, alpha-olefin sulfonates,alkyl naphthalene sulfonates, paraffin sulfonates, lignin sulfonates,alkyl sulfosuccinates, ethoxylated sulfosuccinates, alkyl ethersulfosuccinates, alkylamide sulfosuccinates, alkyl sulfosuccinamate,alkyl sulfoacetates, alkyl phosphates, phosphate ester, alkyl etherphosphates, acyl sarconsinates, acyl isethionates, N-acyl taurates,N-acyl-N-alkyltaurates, alkyl carboxylates, or a combination thereof.

Nonionic Surfactants

Nonionic surfactants are surfactants having no electrical charge whendissolved or dispersed in an aqueous medium. In at least one embodimentof the composition described herein, one or more nonionic surfactantsare used as they provide the desired wetting and emulsification actionsand do not significantly inhibit spore stability and activity. Thenonionic surfactant(s) may be either water soluble nonionic surfactants,water insoluble nonionic surfactants, or a combination of water solublenonionic surfactants and water insoluble nonionic surfactants.

Water Insoluble Nonionic Surfactants

Non-limiting examples of water insoluble nonionic surfactants includealkyl and aryl: glycerol ethers, glycol ethers, ethanolamides,sulfoanylamides, alcohols, amides, alcohol ethoxylates, glycerol esters,glycol esters, ethoxylates of glycerol ester and glycol esters,sugar-based alkyl polyglycosides, polyoxyethylenated fatty acids,alkanolamine condensates, alkanolamides, tertiary acetylenic glycols,polyoxyethylenated mercaptans, carboxylic acid esters,polyoxyethylenated polyoxyproylene glycols, sorbitan fatty esters, orcombinations thereof. Also included are EO/PO block copolymers (EO isethylene oxide, PO is propylene oxide), EO polymers and copolymers,polyamines, and polyvinylpynolidones.

Water Soluble Nonionic Surfactants

Non-limiting examples of water soluble nonionic surfactants includesorbitan fatty acid alcohol ethoxylates and sorbitan fatty acid esterethoxylates.

Combination of Nonionic Surfactants

In one embodiment, the compositions described herein comprise at leastone or more nonionic surfactants. In one embodiment, the compositionscomprise at least one water insoluble nonionic surfactant and at leastone water soluble nonionic surfactant. In still another embodiment, thecompositions comprise a combination of nonionic surfactants havinghydrocarbon chains of substantially the same length.

Other Surfactants

In another embodiment, the compositions described herein may alsocomprise organosilicone surfactants, silicone-based antifoams used assurfactants in silicone-based and mineral-oil based antifoams. In yetanother embodiment, the compositions described herein may also comprisealkali metal salts of fatty acids (e.g., water soluble alkali metalsalts of fatty acids and/or water insoluble alkali metal salts of fattyacids).

Herbicide(s):

In one embodiment, the compositions described herein may furthercomprise one or more herbicides. In a particular embodiment, theherbicide may be a pre-emergent herbicide, a post-emergent herbicide, ora combination thereof.

Suitable herbicides include chemical herbicides, natural herbicides(e.g., bioherbicides, organic herbicides, etc.), or combinationsthereof. Non-limiting examples of suitable herbicides include bentazon,acifluorfen, chlorimuron, lactofen, clomazone, fluazifop, glufosinate,glyphosate, sethoxydim, imazethapyr, imazamox, fomesafe, flumiclorac,imazaquin, clethodim, pendimethalin;3,4-Dimethyl-2,6-dinitro-N-pentan-3-yl-aniline;N-(1-ethylpropyl)-2,6-dinitro-3,4-xylidine; pronamide; propyzamide;3,5-Dichloro-N-(1,1-dimethylpropynyl)benzamide;3,5-Dichloro-N-(1,1-dimethyl-2-propynyl)benzamide;N-(1,1-Dimethylpropynyl)-3,5-dichlorobenzamide; S-ethylN-ethylthiocyclohexanecarbamate; trifluralin;2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline; glyphosate;N-(phosphonomethyl)glycine; and derivatives thereof. In one embodiment,the one or more herbicides for use in accordance with this disclosureinclude pronamide (commercially referred to as Kerb®); propyzamide;3,5-Dichloro-N-(1,1-dimethylpropynyl)benzamide;3,5-Dichloro-N-(1,1-dimethyl-2-propynyl)benzamide;N-(1,1-Dimethylpropynyl)-3,5-dichlorobenzamide; cycloate, S-ethylN-ethylthiocyclohexanecarbamate (commercially referred to as Ro-Neet®);trifluralin; 2,6-Dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline;glyphosate; N-(phosphonomethyl)glycine; and derivatives thereof.Commercial products containing each of these compounds are readilyavailable. Herbicide concentration in the composition will generallycorrespond to the labeled use rate for a particular herbicide.

Fungicide(s):

In one embodiment, the compositions described herein may furthercomprise one or more fungicides. Fungicides useful to the compositionsdescribed herein will suitably exhibit activity against a broad range ofpathogens, including but not limited to Phytophthora, Rhizoctonia,Fusarium, Pythium, Phomopsis or Selerotinia and Phakopsora andcombinations thereof.

Non-limiting examples of commercial fungicides which may be suitable forthe compositions disclosed herein include PROTÉGÉ, RIVAL or ALLEGIANCEFL or LS (Gustafson, Plano, Tex.), WARDEN RTA (Agrilance, St. Paul,Minn.), APRON XL, APRON MAXX RTA or RFC, MAXIM 4FS or XL (Syngenta,Wilmington, Del.), CAPTAN (Arvesta, Guelph, Ontario) and PROTREAT(Nitragin Argentina, Buenos Ares, Argentina). Active ingredients inthese and other commercial fungicides include, but are not limited to,fludioxonil, mefenoxam, azoxystrobin and metalaxyl. Commercialfungicides are most suitably used in accordance with the manufacturer'sinstructions at the recommended concentrations.

Insecticide(s):

In one embodiment, the compositions described herein may furthercomprise one or more insecticides. Insecticides useful to thecompositions described herein will suitably exhibit activity against abroad range of insects including, but not limited to, wireworms,cutworms, grubs, corn rootworm, seed corn maggots, flea beetles, chinchbugs, aphids, leaf beetles, stink bugs, and combinations thereof.

Non-limiting examples of commercial insecticides which may be suitablefor the compositions disclosed herein include CRUISER (Syngenta,Wilmington, Del.), GAUCHO and PONCHO (Gustafson, Plano, Tex.). Activeingredients in these and other commercial insecticides includethiamethoxam, clothianidin, and imidacloprid. Commercial insecticidesare most suitably used in accordance with the manufacturer'sinstructions at the recommended concentrations.

Methods

In another aspect, methods of using the deposited strains andcompositions described herein are disclosed.

In one embodiment a method for enhancing plant growth is described. Themethod comprises contacting a plant or plant part with an inoculum ofone or more bacterial strains selected from the group consisting of:

-   -   the strain having the deposit accession number NRRL B-50608;    -   the strain having the deposit accession number NRRL B-50609;    -   the strain having the deposit accession number NRRL B-50610;    -   the strain having the deposit accession number NRRL B-50611;    -   the strain having the deposit accession number NRRL B-50612; or    -   a mixture of two or more of the strains.

In a particular embodiment, the inoculum may comprise one or more of theabove mentioned deposited strains (e.g., including at least two of theabove strains, at least three of the above strains, at least four of theabove strains, up to and including all of the above strains).

In an embodiment, the inoculum comprises the strain having the depositaccession number NRRL B-50608. In an embodiment, the inoculum comprisesthe strain having the deposit accession number NRRL B-50609. In anembodiment, the inoculum comprises the strain having the depositaccession number NRRL B-50610. In an embodiment, the inoculum comprisesthe strain having the deposit accession number NRRL B-50611. In anembodiment, the inoculum comprises the strain having the depositaccession number NRRL B-50612.

In still another embodiment, the step of contacting a plant or plantpart with an inoculum of one or more of the deposited bacterial strainscomprises contacting a plant or plant part with one or more of thecompositions described herein. The inoculum(s) or compositions may bemade to contact the plant or plant part according to methods known tothose skilled in the art. Non-limiting examples include in-furrowintroduction, coating seeds, etc. In a particular embodiment, thecontacting step comprises in-furrow introduction of the inoculum orcompositions described herein. In a particular embodiment, thecontacting step comprises on-seed (seed coating) introduction of theinoculum or compositions described herein.

In certain embodiments, the step of contacting a plant or plant partwith an inoculum of one or more of the deposited bacterial strainscomprises introducing the inoculum into the soil in an amount of1×10¹-1×10⁸, more preferably 1×10⁶-1×10¹² colony forming units perhectare. In other certain embodiments, the step of contacting a plant orplant part with an inoculum of one or more of the deposited bacterialstrains comprises introducing the deposited bacterial strains as a seedcoated with 1×10¹-1×10⁸, more preferably 1×10²-1×10⁶ colony formingunits per seed.

In another aspect, the method comprises growing plants in a soilcomprising one or more of the bacterial strain. The method comprises:

-   -   a) treating the soil an inoculum of one or more bacterial        strains selected from the group consisting of:        -   the strain having the deposit accession number NRRL B-50608;        -   the strain having the deposit accession number NRRL B-50609;        -   the strain having the deposit accession number NRRL B-50610;        -   the strain having the deposit accession number NRRL B-50611;        -   the strain having the deposit accession number NRRL B-50612;            or a mixture of two or more of the strains; and    -   b) growing a plant in the treated soil.

In a particular embodiment, the inoculum may comprise one or more of theabove mentioned deposited strains (e.g., including at least two of theabove strains, at least three of the above strains, at least four of theabove strains, up to and including all of the above strains).

In an embodiment, the inoculum comprises the strain having the depositaccession number NRRL B-50608. In an embodiment, the inoculum comprisesthe strain having the deposit accession number NRRL B-50609. In anembodiment, the inoculum comprises the strain having the depositaccession number NRRL B-50610. In an embodiment, the inoculum comprisesthe strain having the deposit accession number NRRL B-50611. In anembodiment, the inoculum comprises the strain having the depositaccession number NRRL B-50612.

The step of treating the soil with an inoculum of one or more of thedeposited bacterial strains comprises treating the soil with one or moreof the compositions described herein. The inoculum(s) or compositionsmay be introduced into the soil according to methods known to thoseskilled in the art. Non-limiting examples include in-furrow treatment,coating seeds, etc. In a particular embodiment, the treating stepcomprises in-furrow treatment of the inoculum or compositions describedherein. In a particular embodiment, the treating step comprises on-seed(seed coating)treatment of the inoculum or compositions describedherein.

In a particular embodiment, the step of treating the soil with aninoculum of one or more of the deposited bacterial strains comprisestreating the soil with one or more of the compositions described herein.In certain embodiments, the step of treating the soil with an inoculumof one or more of the deposited bacterial strains comprises treating thesoil with an inoculum in an amount of 1×10¹-1×10⁸, more preferably1×10⁶-1×10¹² colony forming units per hectare. In other certainembodiments, the step of treating the soil with an inoculum of one ormore of the deposited bacterial strains comprises introducing thedeposited bacterial strains as a seed coated with 1×10¹-1×10⁸, morepreferably 1×10²-1×10⁶ colony forming units per seed.

In another embodiment, the method further comprises the step of plantinga plant or plant part. The planting step can occur before, after orduring the treating step. In one embodiment, the planting step occursbefore the treating step. In another embodiment, the planting stepoccurs during the treating step (e.g., the planting step occurssimultaneously with the treating step, the planting step occurssubstantially simultaneous with the treating step, etc.). In stillanother embodiment, the planting step occurs after the treating step.

In another embodiment, the method further comprises the step ofsubjecting the soil to one or more agriculturally beneficial ingredientsdescribed herein. In one embodiment, the step of subjecting the soil toone or more agriculturally beneficial ingredients occurs before, during,after, or simultaneously with the treating step. In one embodiment, thestep of subjecting the soil to one or more agriculturally beneficialingredients as described herein occurs before the treating step. Inanother embodiment, the step of subjecting the soil to one or moreagriculturally beneficial ingredients as described herein occurs duringthe treating step. In still another embodiment, the step of subjectingthe soil to one or more agriculturally beneficial ingredients asdescribed herein occurs after the treating step. In yet anotherembodiment, the step of subjecting the soil to one or moreagriculturally beneficial ingredients as described herein occurssimultaneously with the treating step (e.g., treating the soil with oneor more of the compositions described herein, etc.).

In yet another embodiment, the invention includes a method for treatingseeds comprising applying to the seeds an inoculum of one or morebacterial strains selected from the group consisting of:

-   -   the strain having the deposit accession number NRRL B-50608;    -   the strain having the deposit accession number NRRL B-50609;    -   the strain having the deposit accession number NRRL B-50610;    -   the strain having the deposit accession number NRRL B-50611;    -   the strain having the deposit accession number NRRL B-50612; or        a mixture of two or more of the strains.

In a particular embodiment, the method for treating seeds may compriseone or more of the above mentioned deposited strains (e.g., including atleast two of the above strains, at least three of the above strains, atleast four of the above strains, up to and including all of the abovestrains).

In an embodiment, the method of treating seeds comprises applying to theseed the strain having the deposit accession number NRRL B-50608. In anembodiment, the method of treating seeds comprises applying to the seedthe strain having the deposit accession number NRRL B-50609. In anembodiment, the method of treating seeds comprises applying to the seedthe strain having the deposit accession number NRRL B-50610. In anembodiment, the method of treating seeds comprises applying to the seedthe strain having the deposit accession number NRRL B-50611. In anembodiment, the method of treating seeds comprises applying to the seedthe strain having the deposit accession number NRRL B-50612.

In yet another embodiment, the method further comprises the step ofapplying to the seeds one or more agriculturally beneficial ingredientsto the seed. In another embodiment, the method comprises applying to theseeds any of the compositions described herein to the seeds.

In still another embodiment, the method comprises storing seeds with aninoculum of at least one or more of the isolated bacterial strains in asubstantially moisture free environment for a period of time, e.g., atleast 1 day, at least 2 days, at least 3 days, at least 4 days, at least5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months, atleast 1 year or more. In one aspect of the method, the seeds areleguminous plant seeds. In another aspect, the leguminous plant seedsare soybean seeds.

The methods described herein are potentially useful for improving growthconditions resulting in increased phosphorous uptake and/or yield forany type of plant. In one particular embodiment the plant is selectedfrom the group consisting of non-legumes, legumes, Brassica spp.,cereals, fruits, vegetables, nuts, flowers, and turf. Particularly thecereals are wheat, corn, rice, oat, rye, barley. Particularly legumesare lentil, chickpeas, beans, soybeans, peas, and alfalfa.

In another particular embodiment the plants are selected from the groupconsisting of alfalfa, rice, wheat, barley, rye, oat, cotton, sunflower,peanut, corn, potato, sweet potato, bean, pea, chickpeas, lentil,chicory, lettuce, endive, cabbage, brussel sprout, beet, parsnip,turnip, cauliflower, broccoli, turnip, radish, spinach, onion, garlic,eggplant, pepper, celery, carrot, squash, pumpkin, zucchini, cucumber,apple, pear, melon, citrus, strawberry, grape, raspberry, pineapple,soybean, tobacco, tomato, sorghum, and sugarcane.

Seed Coatings

In another aspect, seeds are coated with one or more bacterial strainsselected from the group consisting of:

-   -   the strain having the deposit accession number NRRL B-50608;    -   the strain having the deposit accession number NRRL B-50609;    -   the strain having the deposit accession number NRRL B-50610;    -   the strain having the deposit accession number NRRL B-50611;    -   the strain having the deposit accession number NRRL B-50612; or    -   a mixture of two or more of the strains.

In a particular embodiment, the seed(s) is coated with one or more ofthe above mentioned deposited strains (e.g., including at least two ofthe above strains, at least three of the above strains, at least four ofthe above strains, up to and including all of the above strains).

In an embodiment, the seed(s) is coated with the strain having thedeposit accession number NRRL B-50608. In an embodiment, the seed(s) iscoated with the strain having the deposit accession number NRRL B-50609.In an embodiment, the seed(s) is coated with the strain having thedeposit accession number NRRL B-50610. In an embodiment, the seed(s) iscoated with the strain having the deposit accession number NRRL B-50611.In an embodiment, the seed(s) is coated with the strain having thedeposit accession number NRRL B-50612.

In one embodiment, seeds may be treated with any of the composition(s)described herein in several ways but preferably via spraying ordripping. Spray and drip treatment may be conducted by formulatingcompositions described herein and spraying or dripping thecomposition(s) onto a seed(s) via a continuous treating system (which iscalibrated to apply treatment at a predefined rate in proportion to thecontinuous flow of seed), such as a drum-type of treater. Batch systems,in which a predetermined batch size of seed and composition(s) asdescribed herein are delivered into a mixer, may also be employed.Systems and apparati for performing these processes are commerciallyavailable from numerous suppliers, e.g., Bayer CropScience (Gustafson).

In another embodiment, the treatment entails coating seeds. One suchprocess involves coating the inside wall of a round container with thecomposition(s) described herein, adding seeds, then rotating thecontainer to cause the seeds to contact the wall and the composition(s),a process known in the art as “container coating”. Seeds can be coatedby combinations of coating methods. Soaking typically entails usingliquid forms of the compositions described. For example, seeds can besoaked for about 1 minute to about 24 hours (e.g., for at least 1 min, 5min, 10 min, 20 min, 40 min, 80 min, 3 hr, 6 hr, 12 hr, 24 hr).

In certain embodiments, a seed(s) coated with one or more of thecompositions described herein will comprise 1×10¹-1×10⁸, more preferably1×10²-1×10⁶ colony forming units of one or more of the depositedbacterial strains per seed.

EXAMPLES

The following examples are provided for illustrative purposes and arenot intended to limit the scope of the invention as claimed herein. Anyvariations in the exemplified examples which occur to the skilledartisan are intended to fall within the scope of the present invention.

Materials & Methods

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty at the Microbial Genomics and Bioprocessing ResearchUnit (NRRL) National Center for Agricultural Utilization Research 1815N. University Street, Peoria, Ill. 61604, USA and given the followingaccession number:

TABLE 1 Deposit of Biological Material Identification Accession NumberDate of Deposit Bradyrhizobia japonicum NRRL B-50612 30 Nov. 2011Bradyrhizobia japonicum NRRL B-50611 30 Nov. 2011 Bradyrhizobiajaponicum NRRL B-50610 30 Nov. 2011 Bradyrhizobia japonicum NRRL B-5060930 Nov. 2011 Bradyrhizobia japonicum NRRL B-50608 30 Nov. 2011

The strains have been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a pure culture of the deposited strain. Thedeposit is available as required by foreign patent laws in countrieswherein counterparts of the subject application or its progeny arefiled. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by governmental action.

Media

TABLE 2 Components of G16 medium. G16 Constituents (g/L Distilleddeionized water (DDW)) g/L Potassium phosphate dibasic K₂HPO₄ 0.550Magnesium sulphate heptahydrate MgSO₄•7H₂O 0.200 Calcium ChlorideDihydrate CaCl₂•2H₂O 0.130 Oxoid yeast extract (LP0021) 0.750 Ammoniumchloride NH₄Cl 0.200 L-Glutamic acid monosodium monohydrate 0.250Sucrose 1.500 Dextrose Anhydrous 4.500 Iron (III) Chloride hexahydrateFeCl₃•6H₂O Stock 0.200 (80 g/L DDW) stored at 4° C. for up to 6 months)Corn steep solids (Sigma) 0.400 Trace Element Stock (see below)* 365 μlVitamin Stock (see below) ** 365 μl pH 6.800

TABLE 3 Trace element Stock Store at 4° C. for up to 6 months.Constituent (g/L DDW) g/L Nickel (II) Chloride Hexahydrate NiCl₂•6H₂00.69 Cupric sulfate pentahydrate CuSO₄•5H₂O 0.22 Boric Acid H₃BO₃ 7.87Manganous sulfate monohydrate MnSO₄•H₂O 5.06 Zinc sulfate heptahydrateZnSO₄•7H₂O 0.61 Sodium molybdateNa2MoO₄•2H₂O 0.61 Cobalt (II)hexahydrate CoCl₂•6H₂O 0.66 *Trace elements are added with all othercomponents before sterilization.

TABLE 4 Vitamin Stock - filter sterilized followed by storage at 4° C.for up to 6 months. Constituent (g/L DDW) g/L Thiamine Hydrochloride1.38 Panthothenic acid 0.55 **Vitamins are added after the media hasbeen sterilized and has cooled, typically at time of inoculation.

TABLE 5 Components of Yeast Extract Mannitol (YEM) Medium. Yeast ExtractMannitol Agar (YEMA) Constituents (g/L DDW) g/L or mL/L Mannitol 10.0 gOxoid Yeast Extract (LP0021) 0.50 g Sodium Chloride NaCl 0.10 g STOCK -Potassium phosphate dibasic K₂HPO₄ 10 mL (50 g/1000 ml) STOCK -Magnesium sulphate heptahydrate MgSO₄•7H₂O 10 mL (20 g/1000 ml) pH 6.8

Example I Determine 99.99% Kill Rate for USDA 532C

The following experiment(s), consisting of three (3) studies, wasperformed to determine the 99.99% kill rate for parental strainBradyrhizobia japonicum USDA 532C.

Parental strain USDA 532C was grown in two 10 ml G16 (Tables 2-4) andYEM (Table 5) disposable culture tubes (VWR, 18×150 mm, #47729-583) fortwo days and harvested to obtain the highest cell concentration. Thiswas achieved by combining both culture tubes into one tube andconcentrating the cells down to 2 ml. Approximately fifty soybean seeds(variety Stine RR 1108-4) were surfaced sterilized in 50 ml sterile,disposable centrifuge tube (Fisher brand, #06-443-18) containing 5%household bleach solution for 30 seconds and rinsed with steriledeionized (DI) water. The sterilization step was repeated for fivetimes. The seeds were immediately placed in a sterilized Petri dish anddried under the laminar hood. Once the seeds were completely dried andtransferred to a 250 ml beaker, 1.5 ml of the concentrated parentalstrain USDA 532C culture was added to the seeds. The seeds were swirledin the beaker to evenly coat the seeds and allowed to dry under thehood. The beaker, containing the seeds, was wrapped with blue,sterilization paper and left in the hood until the experiment wascompleted. Time points were taken at zero time, every two days for oneweek, and every week until complete cell death occurred. Results areprovided in Table 6.

TABLE 6 CFU per seed and percent kill rate for study 1. Number of daysCFU per seed Percent kill rate 0 3.06 × 10⁸ 0.00 3 3.72 × 10⁷ 87.86 74.20 × 10⁶ 98.63 14 3.07 × 10⁶ 99.00 18 6.43 × 10⁵ 99.79 24 2.87 × 10⁵99.91 37 2.64 × 10⁴ 99.99

As shown in Table 6 the initial CFU per seed for parental strain USDA532C was 3.06×10⁸ and at days 37 the CFU was at 2.64×10⁸. The percentkill rate from times 0 to 37 days was calculated to be 99.99%.

The procedure was repeated except G16 was used as the initial growingmedium. The results are provided in Table 7.

TABLE 7 CFU per seed and percent kill rate for study 2. Number of daysCFU per seed Percent kill rate 0 2.01 × 10⁹ 0.00 2 3.13 × 10⁸ 84.41 63.26 × 10⁷ 98.38 10 9.14 × 10⁶ 99.55 16 3.50 × 10⁶ 99.83 22 1.40 × 10⁶99.93 29 3.38 × 10⁵ 99.98 37 6.23 × 10⁴ 100.00

As shown in Table 7, when G16 was used as the initial growing medium ittook from 29 to 37 days for the kill rate to reach 99.99%.

A third desiccation study was completed to determine if G16 and YEMmedia affected the rate of desiccation of parental strain USDA 532C. Theresults are provided in Tables 8 and 9 respectively.

TABLE 8 CFU per seed and percent kill rate for parental strain USDA 532Cgrown G16 medium. Number of days CFU per seed Percent kill rate 0 2.95 ×10⁹ 0.00 2 8.69 × 10⁷ 97.06 7 2.93 × 10⁷ 99.01 14 1.68 × 10⁷ 99.43 212.11 × 10⁶ 99.93 28 7.50 × 10⁴ 100.00

TABLE 9 CFU per seed and percent kill rate for parental strain USDA 532Cgrown YEM medium. Number of days CFU per seed Percent kill rate 0 2.90 ×10⁸ 0.00 2 3.46 × 10⁶ 98.81 7 1.81 × 10⁶ 99.38 14 1.31 × 10⁶ 99.55 217.33 × 10⁵ 99.75 28 7.00 × 10⁴ 99.98

As shown in Tables 8 and 9, there was no difference in the desiccationrate for parental strain USDA 532C when grown in G16 or YEM. The 99.99%kill rate was observed for the third study at approximately 28 dayswhich is similar to what was observed in studies one (1) and two (2)supra.

Example II Determine the Kill Rate of USDA 532C Using EthylMethanesulfonate (EMS)

The following experiment(s) were performed to determine the applicationrate of the mutagen, ethyl methanosulfonate (EMS) that would give a99.9-99.99 percent kill rate for parental strain USDA 532C. This ratedetermination will become part of the mutagenesis protocol used togenerate desiccation-resistant putative mutants although the method ofmutagenesis may evolve for efficiency.

Inocula Preparation:

Parental strain USDA 532C was grown in six 10 ml YEM disposable culturetubes for two days and 5 ml of the culture was inoculated into four 250ml flasks containing 50 ml YEM medium. The flasks were incubated for twodays at 30° C. shaker. The culture from the flasks were subsequentlycentrifuged in 50 ml disposable sterile tubes at 8,000 rpm for tenminutes in a Sorvall RC 6 Plus® centrifuge and combined into one tube.The pellet was re-suspended in 4 ml of fresh YEM medium and separatedinto four 1.5 ml microcentrifuge tubes. The tubes would each representdifferent application rates used for the mutagenesis process.

Mutagenesis Process:

Once the culture have been aliquoted into separate tubes and the mutagenEMS (Sigma, C3H8O3S, FW 124.16, #M0880-1G) added to each tube, the tubeswere vortexed vigorously and placed in an empty 250 ml flask. The flaskcontaining the reaction tubes were incubated for 30 minutes at 30° C. ina shaker. Immediately following the incubation period, the tubes werewashed five times with 0.16M sodium thiosulfate (STS, Fisher Chemical,Na2S2O3*5H2O, FW 248.18, #S445-3)) solution to inactivate the mutagen.After washing, the cells in the reaction tubes were sheared with 21gauge syringe needle (BD 1 ml 21G1 Latex Free Syringe PrecisionGlide®Needle, 0.8 mm×25 mm, #309624) and dilutions were completed and platedon YEMA plates. The cell counts were available after five daysincubation at 30° C. and the percent kill of the EMS application ratewas calculated. To calculate the percent kill rate for each applicationrate, the following equation was used: ([cell count 0 μl EMS(control)−(cell count of μl EMS (treatment))÷cell count 0 μl EMS(control)]×100%). All other experiments following this experiment usedthis equation to calculate the percent kill rate. Results are providedin Table 10.

TABLE 10 Initial rates of EMS to determine upper limit of mutagenesisfor parental strain USDA 532C Application Rate Cell counts (approximatecfu/ml)   0 μl EMS (control) 10⁸ cfu/ml   1 μl EMS similar results to 0μl EMS 10⁷ cfu/ml  10 μl EMS similar results to 1 μl EMS 100 μl EMS 100%kill rate

As shown in Table 10, the initial rates of EMS used were 0 μl, 1 μl, 10μl and 100 μl. There was no difference among 0 μl, 1 μl, and 10 μl EMS,but 100 μl EMS resulted in 100% kill.

Experiments were repeated and refined to determine the acceptable killrate. See Tables 11-17.

TABLE 11 Refining the rates of EMS to determine 99.9% kill rate forparental strain USDA 532C Cell counts Application Rate (approximatecfu/ml) Percent kill rate  0 μl EMS (control) 2.49 × 10⁹ cfu/ml 15 μlEMS 2.24 × 10⁹ cfu/ml 10.17% 25 μl EMS 1.72 × 10⁹ cfu/ml 30.92% 50 μlEMS 1.67 × 10¹ cfu/ml  100%

From the initial finding, the amount of EMS used was narrowed to 0 μl,15 μl, 25 μl and 50 μl EMS. As shown in Table 11, the percent kill ratewas 10.17% to 30.92% for 15 μl and 25 μl EMS applications and 100% for50 μl.

TABLE 12 Additional refinement of the application rates of EMS forparental strain USDA 532C Cell counts Application Rate (approximatecfu/ml) Percent kill rate  0 μl EMS (control) 4.70 × 10⁹ cfu/ml 25 μlEMS 1.27 × 10⁹ cfu/ml 73.09% 35 μl EMS 5.37 × 10⁸ cfu/ml 88.58% 50 μlEMS 2.93 × 10² cfu/ml  100%

It was determined from Table 11 that 25 μl EMS was still too low of akill rate. The application amounts for Table 12 were 0 μl, 25 μl, 35 μl,and 50 μl EMS. The application at 25 μl EMS had a higher kill rate thanthe results in Table 11 due to the decreased washing; therefore, thekill rate was higher than expected. However, the kill rate was still toolow at 88.58% kill even when 35 μl EMS was used. See Table 12.

TABLE 13 Additional refinement of the application rates of EMS forparental strain USDA 532C Cell counts Application Rate (approximatecfu/ml) Percent kill rate  0 μl EMS (control) 3.93 × 10⁹ cfu/ml 40 μlEMS 6.97 × 10⁸ cfu/ml 82.26% 45 μl EMS 8.57 × 10⁷ cfu/ml 97.82% 50 μlEMS 2.65 × 10⁵ cfu/ml 99.99%

The amount of EMS used was increased to 40 μl, 45 μl, and 50 μl EMS. TheEMS dose rates resulted in percent kill ranges of 82.26%-99.99%. SeeTable 13.

TABLE 14 Repeat application rate used in Table 13. Cell countsApplication Rate (approximate cfu/ml) Percent kill rate  0 μl EMS(control) 7.07 × 10¹¹ cfu/ml  40 μl EMS 1.35 × 10⁹ cfu/ml 99.81% 45 μlEMS 5.27 × 10⁸ cfu/ml 99.93% 50 μl EMS 2.19 × 10⁶ cfu/ml  100%

The results provided in Table 13 were repeated again in Table 14 usingthe same EMS rates and this time the percent kill was 99.81% for 40 μlEMS and 99.93% for 45 μl EMS and 100% for 50 μl EMS. The desired killrate of 99.9% was observed when 45 μl EMS was used so the applicationwill be repeated. See Table 15.

TABLE 15 Repeat application rate used in Table 14. Cell countsApplication Rate (approximate cfu/ml) Percent kill rate  0 μl EMS(control) 1.51 × 10⁸ cfu/ml 45 μl EMS 3.33 × 10⁴ cfu/ml 99.98% 50 μl EMS4.33 × 10¹ cfu/ml  100%

The application rate of 45 μl EMS was duplicated to determine if theresults in Table 14 were repeatable. The kill rate for 45 μl EMS was99.98%. See Table 15.

Example III Mutagenesis

The following experiment(s) were performed to generate putativedesiccation-resistant mutants of strains parental strain USDA 532C usingclassical, e.g., chemical, mutagenesis.

Inocula Preparation:

A cell suspension of parental strain USDA 532C was made by taking a loopof cells from a fresh plate of USDA 532C by using a 10 ul sterileplastic loop (Fisher brand, #22-363-600) and mixing the cells in 1 mlsterilized, deionized (DI) water in 1.5 ml disposable microcentrifugetube. The cell suspension was inoculated into two 250 ml flaskscontaining 50 ml YEM medium to achieve a final optical density (OD)OD_(600nm) of 0.01. The flasks were incubated at 30° C. for three daysand the cultures of the two flasks were combined. The culture wascentrifuged for twenty minutes at 8,000 rpm in a Sorvall RC 6 Pluscentrifuge. The supernatant was discarded and the pellet fromre-suspended in 30 ml DI water. OD was taken of the concentrated cultureand was inoculated into ten 250 ml flasks containing 50 ml YEM medium atOD=0.05. These flasks were incubated at 30° C. shaker for two days priorto the culture being used for mutagenesis.

Mutagenesis Process:

The cultures from the ten flasks were combined into a 1 L centrifugebottle. Optical density of the combined cultures was recorded and thecultures were centrifuged for 20 minutes at 8,000 rpm in the Sorvall RC6 Plus® centrifuge. The majority of the supernatant was discardedleaving approximately 30 ml of the supernatant in the centrifuge bottle.The supernatant was mixed with the pellet and transferred into a 50 mlsterile disposable centrifuge tube. The OD of the concentrated culturewas taken and recorded. 1 ml of the concentrated culture was placed intosix 1.5 ml disposable microcentrifuge tubes. The microcentrifuge tubeswere centrifuged and the supernatant was discarded. This was repeatedthree more times or until the size of the pellet had reach the 0.1 mlmark on the microcentrifuge tube. The cells were mixed well with 1 mlfresh YEM medium using a sterile 1 ml 21 gauge syringe needle prior tothe addition of the mutagen, ethyl methanesulfonate (EMS). The rates ofmutagen added to each tube contained a high and a low dose with mediumdosages between the high and low doses as indicated in Experiment II.

Immediately after the addition of EMS, the reaction tubes were placed inan empty 250 ml flask and incubated at 30° C. for 30 minutes. Afterincubation, the reaction tubes were centrifuged for one minute at 13,200rpm using the Eppendorf Centrifuge 5415D. The supernatant of thereaction tubes was discarded. The mutagen in the reaction tubes wasinactivated by washing five times with 1 ml of 0.16M sodium thiosulfate(STS) and mixed vigorously by vortexing the tubes. For each wash cycle,the reaction tubes were centrifuged after vortexing and the supernatantwas discarded. After the fifth time, the reaction tubes were allcombined into one 15 ml disposable tube for use in the enrichmentprocess.

Example IV Enrichment and Desiccation

The following experiment(s) were performed to enrich and desiccate themutated cells of parental strain USDA 532C to eliminate wild typeescapes and increase the putative mutant population and make it easierto isolate the mutant(s) that have desiccation-resistantcharacteristics.

Parental strain USDA 532C was subjected to the mutagenesis processmentioned in Example III. The mutated population of parental strain USDA532C was enriched by inoculating 0.5 ml of the reaction into two 50 mlYEM flasks and incubating the cells for two days at 30° C. shaker. Aftertwo days, the cultures were desiccated by coating the cells onto soybeanseeds and membrane filters and subjected to drying conditions. Theculture from the enrichment step was adjusted to OD_(600nm) of 0.5before it was used to coat both soybean seeds and membrane filters.

Coating of Soybean Seeds:

Forty sterilized soybean seeds were coated with 0.5 ml of the culture.The seeds were placed in a 100 ml sterile beaker to dry under thelaminar hood and covered with autoclave paper. Triplicate samples of theseeds were taken to get an initial CFU of the seeds. For each sample,three seeds were placed in a 15 ml disposable tube containing 5 mlsterile DI water and allowed to expand in the tube for approximately twohours before the suspension was serially diluted and spread onto YEMAplates. The remaining seeds left in the covered, beaker was placed underthe hood for four days before the seeds were enriched.

To enrich the seeds, twenty seeds were placed into a 250 ml flaskcontaining fresh 50 ml YEM. A final CFU was also taken when the seedswere enriched to determine the percent kill rate for the cellpopulation. The same sampling was completed for the second time point asthe initial time point. After the culture containing the seeds wasincubated for two days, the culture was harvested by removing any seeddebris by allowing the debris to settle before removing the supernatant.The supernatant of the culture was centrifuged and the pellet washedwith sterile DI water prior to the culture being used to coat new setsof soybean seeds. This process was repeated until the calculated percentkill rate of the cell culture was less than 80%. Once 80% was achieved,the cell population was ready for isolation of the putative mutants forconfirmation experiment.

Coating of Membrane Filters:

To control for soybean seed inconsistency and contamination issues,membrane filters were used as an alternative medium for cell coating.For membrane filters, 1 ml of the culture was used to coat both durapore(Millipore, 0.22 μm, PVDF, #GVWP02500) and isopore (Millipore, 0.4 μm,polycarbonate, # HTTP02500) membrane filters. For each type of filter,fifteen filters were coated by using a 25 mm Easy Pressure SyringeFilter Holder (VWR, #28144-109) and the filters were placed in sterilePetri dish containing two pieces of sterile, qualitative 125 mm Whatmanpaper (Whatman, #1001125). Once the filters were dried under the laminarhood, triplicates initial CFU were taken for each type of filter byplacing one filter into a 15 ml disposable tube containing 5 ml sterileDI water and mixed by vortexing. After two hours soaking in the 15 mltube, the filter suspension was diluted and plated onto YEMA plates.After the filters have dried under the hood for three days, eightfilters were added into 250 ml flask with 50 ml fresh YEM and incubatedat 30° C. for three days.

A final CFU was taken at the same time the filters were enriched to getthe percent kill rate calculation. The same method was used for thefinal CFU as the initial CFU. The process of coating and drying wasrepeated until the percent kill rate was less than 80%. After 80% wasachieved, single colony isolates were selected for further confirmation.

Example V Confirmation of Putative Mutants

The following experiment(s) were to confirm the putativedesiccation-resistant mutants by comparing their on-seed survivabilityafter seed application with the original parent strain Bradyrhizobiumjaponicum strain USDA 532C.

When 80% kill rate was observed for the seeds or filters, singlecolonies were randomly picked from the final time point and the putativemutants of a set of mutagenesis were analyzed for desiccation tolerancecharacteristic. The twenty single colonies picked from each set ofmutagenesis results were individually grown in 250 ml flask containing50 ml YEM medium. Each putative mutant strain was incubated at 30° C.shaker for three days and OD for each strain was adjusted to 0.5. Eachstrain was used to coat thirty unsterile soybean seeds with 0.5 mlculture in a 100 ml beaker covered by autoclave paper. Time points weretaken at T=0, T=3, and T=7 days for the first round. Triplicate seedsamplings were taken for each time point where each sample consisted ofthree seeds placed in 5 ml sterile DI water in 15 ml disposable tube.The seeds were allowed to expand for two hours before each sample wasdiluted and plated onto YEMA plates. After comparing the amount of cellsrecovered from each time point in relation to parental strain USDA 532C,any strain that performed better than the parent strain was subjected toa second round of confirmation. See FIG. 1. For the second round, thestrains that had the best desiccation tolerance compared to the wildtype were tested for desiccation again. See FIG. 2. Time points weretaken at T=0, T=7, and T=14 days. The set of putative mutants from thesecond round was further confirmed for desiccation tolerance two moretimes. See FIGS. 3-6.

Twenty putative mutants of parental strain USDA 532C were isolated andscreened for desiccation resistance. See FIGS. 1-3. Of the twentyputative strains tested, five putative mutant strains were confirmed fortheir desiccation tolerance characteristics when compared to thedesiccation tolerance of parental strain, Bradyrhizobium japonicumstrain USDA 532C. See FIGS. 4-6.

Example VI Greenhouse Testing of Confirmed Mutant Strains

The following experiment(s) were performed to test the putative mutantstrains in the greenhouse to test the performance of the mutant strainsagainst the performance of the parent strain, USDA 532C.

The mutant strains having the best desiccation tolerance compared to theparent strain USDA 532C were tested in the greenhouse for performanceagainst parental strain USDA 532C. The mutant and parent strains weregrown in 50 ml YEM for two days before seed coating. Each strain wasplanted three different times; T=0, T=7, and T=14 days after seedcoating. All time points were set up at the same time but the seeds wereplanted at the specified times. To set up for the time points, thirtysoybean seeds were coated with 0.5 ml of culture at OD_(600nm)=0.5 in100 ml beaker and the T=0 day time point was allowed to sit under thehood for 30 minutes before planting. The other two time points wereallowed to dry completely and covered with autoclave paper. The seedsfrom the last two time points were planted at a later date. At each timepoint, two seeds were planted per pot for ten pots per strain. Theleftover seeds were used to take a CFU for comparison with T=0. Afternine weeks of growing in the greenhouse, the soybean pods were harvestedfrom each plant from each time point and the dry weights were analyzedfor statistical significance.

When the soybean pod weights of the mutant strains were compared to theparent strain at any of time points, there was no statisticalsignificance at 95% confidence. This indicates that there was noperformance difference between the mutant strains and parent strain thatwould affect the production of soybean pods when the mutant strains wereused to coat soybean seeds.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

The invention claimed is:
 1. A method of treating seeds comprisingapplying to said seeds an inoculum that comprises at least onemutagenized Bradyrhizobium japonicum strain selected from the groupconsisting of: the strain having the deposit accession number NRRLB-50612; the strain having the deposit accession number NRRL B-50611;the strain having the deposit accession number NRRL B-50610; the strainhaving the deposit accession number NRRL B-50609; and the strain havingthe deposit accession number NRRL B-50608.
 2. The method of claim 1,wherein said inoculum further comprises one or more plant signalmolecules.
 3. The method of claim 1, wherein said inoculum furthercomprises a lipo-chitooligosaccharide (LCO).
 4. The method of claim 3,wherein said LCO is synthetic.
 5. The method of claim 3, wherein saidLCO is recombinant.
 6. The method of claim 3, wherein said LCO isnaturally occurring.
 7. The method of claim 3, wherein said LCO isobtained from a species of Rhizobia selected from the group consistingof Rhizobium spp., Sinorhizobium spp., and Azorhizobium spp.
 8. Themethod of claim 3, wherein said LCO is obtained from Bradyrhizobiumjaponicum.
 9. The method of claim 3, wherein said LCO is obtained froman arbuscular mycorrhizal fungus.
 10. The method of claim 1, whereinsaid inoculum further comprises a chitinous compound.
 11. The method ofclaim 10, wherein said chitinous compound is a chito-oligomer (CO). 12.The method of claim 11, wherein said CO is synthetic.
 13. The method ofclaim 11, wherein said CO is recombinant.
 14. The method of claim 11,wherein said CO is naturally occurring.
 15. The method of claim 1,wherein said inoculum further comprises a flavonoid.
 16. The method ofclaim 15, wherein said flavonoid is selected from the group consistingof luteolin, apigenin, tangeritin, quercetin, kaempferol, myricetin,fisetin, isorhamnetin, pachypodol, rhamnazin, hesperetin, naringenin,formononetin, eriodictyol, homoeriodictyol, taxifolin, dihydroquercetin,dihydrokaempferol, genistein, daidzein, glycitein, catechin,gallocatechin, catechin 3-gallate, gallocatechin 3-gallate, epicatechin,epigallocatechin, epicatechin 3-gallate, epigallocatechin 3-gallate,cyanidin, delphinidin, malvidin, pelargonidin, peonidin, petunidin, andderivatives thereof.
 17. The method of claim 1, wherein said inoculumfurther comprises jasmonic acid or a derivative thereof.
 18. The methodof claim 1, wherein said inoculum further comprises linoleic acid or aderivative thereof.
 19. The method of claim 1, wherein said inoculumfurther comprises linolenic acid or a derivative thereof.
 20. The methodof claim 1, wherein said inoculum further comprises a karrikin.
 21. Themethod of claim 1, wherein said inoculum further comprises a herbicide,an insecticide and/or a fungicide.
 22. The method of claim 1, whereinsaid inoculum further comprises at least one phosphate-solubilizingmicroorganism.
 23. The method of claim 22, wherein said at least onephosphate-solubilizing microorganism comprises a strain of Penicillium.24. The method of claim 22, wherein said at least onephosphate-solubilizing microorganism comprises a strain of P. bilaiae.25. The method of claim 24, wherein said strain of P. bilaiae isselected from the group consisting of the strain having the depositaccession number NRRL 50162, the strain having the deposit accessionnumber NRRL 50169, the strain having the deposit accession number ATCC20851, the strain having the deposit accession number ATCC 22348, andthe strain having the deposit accession number ATCC
 18309. 26. Themethod of claim 22, wherein said at least one phosphate-solubilizingmicroorganism comprises a strain of P. gaestrivorus.
 27. The method ofclaim 26, wherein said strain of P. gaestrivorus is the strain havingthe deposit accession number NRRL
 50170. 28. The method of claim 1,further comprising the step of storing treated seeds in a substantiallymoisture free environment for a period of at least 1 month, at least 2months, at least 3 months, at least 4 months, at least 5 months, atleast 6 months, at least 1 year or more.
 29. The method of claim 1,wherein said seeds are leguminous plant seeds.
 30. The method of claim29, wherein said leguminous plant seeds are soybean seeds.